Functional characterization and evaluation of protective efficacy of EA752-862 monoclonal antibody against B. anthracis vegetative cell and spores.

The most promising means of controlling anthrax, a lethal zoonotic disease during the early infection stages, entail restricting the resilient infectious form, i.e., the spores from proliferating to replicating bacilli in the host. The extractible antigen (EA1), a major S-layer protein present on the vegetative cells and spores of Bacillus anthracis, is highly immunogenic and protects mice against lethal challenge upon immunization. In the present study, mice were immunized with r-EA1C, the C terminal crystallization domain of EA1, to generate a neutralizing monoclonal antibody EA752-862, that was evaluated for its anti-spore and anti-bacterial properties. The monoclonal antibody EA752-862 had a minimum inhibitory concentration of 0.08 mg/ml, was bactericidal at a concentration of 0.1 mg/ml and resulted in 100% survival of mice against challenge with B. anthracis vegetative cells. Bacterial cell lysis as observed by scanning electron microscopy and nucleic acid leakage assay could be attributed as a possible mechanism for the bactericidal property. The association of mAb EA752-862 with spores inhibits their subsequent germination to vegetative cells in vitro, enhances phagocytosis of the spores and killing of the vegetative cells within the macrophage, and subsequently resulted in 90% survival of mice upon B. anthracis Ames spore challenge. Therefore, owing to its anti-spore and bactericidal properties, the present study demonstrates mAb EA752-862 as an efficient neutralizing antibody that hinders the establishment of early infection before massive multiplication and toxin release takes place.

A Bivalent Protein r-PAbxpB Comprising PA Domain IV and Exosporium Protein BxpB Confers Protection Against B. anthracis Spores and Toxin.

Anthrax vaccines primarily relying only on protective antigen (PA), the cell binding component in anthrax toxins provide incomplete protection when challenged with spores of virulent encapsulated Bacillus anthracis strains. Alternatively, formaldehyde inactivated spores (FIS) or recombinant spore components generate anti-spore immune responses that inhibit the early stages of infection and augment the PA protective efficacy. In the present study domain IV of PA was spliced with exosporium antigen BxpB via a flexible G4S linker to generate a single functional antigen r-PAbxpB that was further assessed for its protective efficacy against anthrax toxins and spore infection. Immunization of mice with r-PAbxpB elicited significantly high titer antibodies comprising IgG1:IgG2a isotypes in 1:1 ratio, balanced up-regulation of both Th1 (IL2, IL12, IFN-γ) and Th2 (IL4, IL5, IL10) cytokines and high frequencies of CD4+ and CD8+ T cell subsets. The anti-r-PAbxpB antibodies significantly enhanced spore phagocytosis, and killing within macrophages; inhibited their germination to vegetative cells and completely neutralized the anthrax toxins as evidenced by the 100% protection in passive transfer studies. Active immunization with r-PAbxpB provided 100 and 83.3% protection in mice I.P. challenged with 5 × LD100 LD of toxins and 5 × 104 cfu/ml Ames spores, respectively while the sham immunized group succumbed to infection in 48 h. Therefore, the ability of r-PAbxpB to generate protective immune responses against both spores and toxin and provide significant protection suggests it as an efficient vaccine candidate against B. anthracis infection.

Recombinant outer membrane protein 25c from Brucella abortus induces Th1 and Th2 mediated protection against Brucella abortus infection in mouse model.

Development of a safe and efficacious vaccine for brucellosis is a long standing challenge for scientists. Recognizing potential antigens towards developing vaccine candidate is crucial. Omp25c, a porin protein of Brucella, is a paralog of two previously identified promising vaccine candidates namely, Omp25 and Omp31, with notable sequence identity. Also, Omp25c is conserved in all major Brucella species. This highlights the possibility of employing this protein in multivalent subunit vaccine based approach of Brucella management. In this study, we were interested in examining the immunogenicity and protective efficacy of Omp25c against Brucella infections. Recombinant unlipidated form of this antigen (rOmp25c) produced, upon intraperitoneal immunization in BALB/c mice along with Freund's adjuvant, was confirmed to be highly immunogenic; leading to high IgG antibody titers during the study duration. The IgG2a/IgG2b ratio of anti-rOmp25c antibodies revealed elicitation of Th2 based humoral immunity. Lymphocyte proliferation study divulged induction of specific memory response and secretion of both Th1-type (IFN-γ, GM-CSF and TNF-α) and Th2-type cytokine (IL-5) from restimulated splenocytes of rOmp25c immunized mice. CD4 T-cell subpopulation was comparatively increased than total B cell subpopulation in case of immunized mice, indicating the induction of strong cell-mediated (Th1 biased) immunity than humoral (Th2) immunity. The collective Th1 plus Th2 immune response specific to rOmp25c could be the reason for protection against Brucella challenge observed in mice groups that was comparable with S19 vaccine strain. Thus, the study encourages rOmp25c as a potent candidate vaccine against brucellosis.

Immunization with recombinant bivalent chimera r-Cpae confers protection against alpha toxin and enterotoxin of Clostridium perfringens type A in murine model.

Clostridium perfringens type A, an anaerobic pathogen is the most potent cause of soft tissue infections like gas gangrene and enteric diseases like food poisoning and enteritis. The disease manifestations are mediated via two important exotoxins, viz. myonecrotic alpha toxin (αC) and enterotoxin (CPE). In the present study, we synthesized a bivalent chimeric protein r-Cpae comprising C-terminal binding regions of αC and CPE using structural vaccinology rationale and assessed its protective efficacy against both alpha toxin (αC) and enterotoxin (CPE) respectively, in murine model. Active immunization of mice with r-Cpae generated high circulating serum IgG (systemic), significantly increased intestinal mucosal s-IgA antibody titres and resulted in substantial protection to the immunized animals (100% and 75% survival) with reduced tissue morbidity when administered with 5×LD(100) doses of αC (intramuscular) and CPE (intra-gastric gavage) respectively. Mouse RBCs and Caco-2 cells incubated with a mixture of anti-r-Cpae antibodies and αC and CPE respectively, illustrated significantly higher protection against the respective toxins. Passive immunization of mice with a similar mixture resulted in 91-100% survival at the end of the 15 days observation period while mice immunized with a concoction of sham sera and respective toxins died within 2-3 days. This work demonstrates the efficacy of the rationally designed r-Cpae chimeric protein as a potential sub unit vaccine candidate against αC and CPE of C. perfringens type A toxemia.

Heterologous protection against alpha toxins of Clostridium perfringens and Staphylococcus aureus induced by binding domain recombinant chimeric protein.

Clostridium perfringens and Staphylococcus aureus are the two important bacteria frequently associated with majority of the soft tissue infections. The severity and progression of the diseases caused by these pathogens are attributed primarily to the alpha toxins they produce. Previously, we synthesized a non-toxic chimeric molecule r-αCS encompassing the binding domains of C. perfringens and S. aureus alpha toxins and demonstrated that the r-αCS hyperimmune polysera reacts with both the native wild type toxins. In the present report, we evaluated efficacy of r-αCS in conferring protection against C. perfringens and S. aureus alpha toxin infections in murine model. Immunization of BALB/c with r-αCS was effective in inducing both high titers of serum anti-r-αCS antibodies after three administrations. Sub-typing the antibody pool revealed high proportions of IgG1 indicating a Th2-polarized immune response. The r-αCS stimulated the proliferation of splenocytes from the immunized mice upon re-induction by the antigen, in vitro. The levels of interleukin-10 increased while TNF-α was found to be downregulated in the r-αCS induced splenocytes. Mice immunized with r-αCS were protected against intramuscular challenge with 5×LD100 doses of C. perfringens and S. aureus alpha toxins with >80% survival, which killed control animals within 48-72h. Passive immunization of mice with anti-r-αCS serum resulted in 50-80% survival. Our results indicate that r-αCS is a remarkable antigen with protective efficacy against alpha toxin mediated C. perfringens and S. aureus soft tissue co-infections.

Functional characterization and evaluation of in vitro protective efficacy of murine monoclonal antibodies BURK24 and BURK37 against Burkholderia pseudomallei.

Burkholderia pseudomallei, the causative agent of melioidosis has been recognized by CDC as a category B select agent. Although substantial efforts have been made for development of vaccine molecules against the pathogen, significant hurdles still remain. With no licensed vaccines available and high relapse rate of the disease, there is a pressing need for development of alternate protection strategies. Antibody-mediated passive protection is promising in this regard and our primary interest was to unravel this frontier of specific mAbs against Burkholderia pseudomallei infections, as functional characterization of antibodies is a pre-requisite to demonstrate them as protective molecules. To achieve this, we designed our study on in vitro-based approach and assessed two mAbs, namely BURK24 and BURK37, reactive with outer membrane proteins and lipopolysaccharide of the pathogen respectively, for their ability to manifest inhibitory effects on the pathogenesis mechanisms of B. pseudomallei including biofilm formation, invasion and induction of apoptosis. The experiments were performed using B. pseudomallei standard strain NCTC 10274 and a clinical isolate, B. pseudomallei 621 recovered from a septicemia patient with diabetic ailment. The growth kinetic studies of the pathogen in presence of various concentrations of each individual mAb revealed their anti-bacterial properties. Minimal inhibitory concentration and minimal bactericidal concentration of both the mAbs were determined by using standards of Clinical and Laboratory Standards Institute (CLSI) and experiments were performed using individual mAbs at their respective bacteriostatic concentration. As an outcome, both mAbs exhibited significant anti-Burkholderia pseudomallei properties. They limited the formation of biofilm by the bacterium and completely crippled its invasion into human alveolar adenocarcinoma epithelial cells. Also, the mAbs were appreciably successful in preventing the bacterium to induce apoptosis in A549 cells. The present study design revealed the protection attributes possessed by BURK24 and BURK37 that has to be further substantiated by additional in vivo studies.

Protective antigen and extractable antigen 1 based chimeric protein confers protection against Bacillus anthracis in mouse model.

Recombinant bivalent chimeric protein was generated comprising of domain 4 of protective antigen (PA4) and carboxy terminal region of extractable antigen 1 (EA1C) by overlap extension PCR. The immunogenicity and protective efficacy of recombinant chimeric protein (PE) and protein mixture (PAEA) along with the individual components, PA4 and EA1C were evaluated in this study. We found that PE and PAEA exhibited higher endpoint titer and elevated IgG1 response. Compared to PA4 and EA1C, the chimeric protein PE and protein mixture PAEA exhibited 1.52 and 1.39 times more proliferative effect on lymphocytes in vitro. The spore uptake by anti-PE and anti-PAEA antibodies was significantly more than the individual components. We further evaluated the effects of antisera on the toxins in vitro and in vivo. Anti-PE and anti-PAEA antibodies displayed nearly 80% protection against crude toxin activity on RAW 264.7 cell lines. We further demonstrated that the anti-PE and anti-PAEA antibodies displayed better protection in controlling the edema induced by crude toxin. Passive immunization with anti-PE and anti-PAEA provided protection against toxin challenge in mice. The present study reveals that the chimeric protein consisting of heterologous regions of PA and EA1 can render better protection than PA4 or EA1C alone against toxins and bacilli.

A recombinant bivalent fusion protein rVE confers active and passive protection against Yersinia enterocolitica infection in mice.

In the present study, a bivalent chimeric protein rVE comprising immunologically active domains of Yersinia pestis LcrV and YopE was assessed for its prophylactic abilities against Yersinia enterocolitica O:8 infection in murine model. Mice immunized with rVE elicited significantly higher antibody titers with substantial contribution from the rV component (3:1 ratio). Robust and significant resistance to Y. enterocolitica infection with 100% survival (P<0.001) was seen in rVE vaccinated mice when intra peritoneal (I.P.) challenged with 10(8)CFU of Y. enterocolitica O:8 against the 75%, 60% and 75% survival seen in mice immunized with rV, rE, rV+rE, respectively. Macrophage monolayer supplemented with anti-rVE polysera illustrated efficient protection (89.41% survival) against challenge of Y. enterocolitica O:8. In contrast to sera from sham-immunized mice, immunization with anti-rVE polysera provided complete protection to BALB/c mice against I.P. challenge with 10(8)CFU of Y. enterocolitica O:8 and developed no conspicuous signs of infection in necropsy. The histopathological analysis of microtome sections confirmed significantly reduced lesion size or no lesion in liver and intestine upon infection in anti-rVE immunized mice. The findings from this study demonstrated the fusion protein rVE as a potential candidate subunit vaccine and showed the functional role of antibodies in protection against Y. enterocolitica infections.